Bringing mechanobiology to the benchtop with single-molecule centrifugation

描述（由申请人提供）：从控制免疫应答中白细胞的粘附和募集到决定细胞命运和组织发育，机械力在整个生物学过程中发挥着关键的调节作用。这个新兴的“机械生物学”领域正在导致对出血性疾病，癌症，骨质疏松症和哮喘等疾病的新理解，因为我们认识到力学可以在分子，细胞和器官水平的生理反应中发挥重要作用。能够精确操纵单个分子和细胞的技术发展（例如光学镊子和AFM）一直是该领域发展的驱动力。然而，该领域的发展受到这种技术的有限使用的阻碍，因为它可能是昂贵的，技术上具有挑战性的，并且产量低。为了克服这些挑战，我们将开发一种大胆的方法，以单分子精度将受控力应用于微观样品，这种方法价格低廉，使用简单，高通量。通过集成离心机和显微镜，我们已经展示了一个原型微力显微镜（CFM）并行执行数千个单分子力实验。我们建议将单分子离心的概念提升到一个新的水平，开发一种功能强大的多用途台式机械生物学仪器，可供各种生物医学研究人员使用。我们将通过以下方式实现这一目标：1）通过将高分辨率成像集成到CFM中，实现结构转变（如蛋白质解折叠）的大规模并行单分子测量; 2）通过将高分辨率CFM直接集成到标准台式离心机中，增加单分子操作技术的可及性; 3）通过在单分子和单细胞水平上测量分子内和分子间的键强度来证明该CFM的多功能性。总之，该项目将产生一个可访问的，高通量和高分辨率的新平台，用于在纳米级的机械力下测量生物系统，具有广泛的应用范围，从测量单个分子内的结构转变，到测量单细胞的亲和力，再到测量软样品的顺应性。该仪器将通过利用实验室设备中最常见的一种-台式离心机，大大降低成本，提高性能和安全性。这种方法将显著降低研究人员进行单分子操纵实验的障碍，因为它只需要很少的技术专长，并且与许多其他方法相比，效率提高了1000倍，成本提高了10-100倍。该项目意义重大，因为它将向许多新的研究人员和系统开放机械生物学和单分子操纵领域，加快发现的步伐。

DESCRIPTION (provided by applicant): From governing the adhesion and recruitment of leukocytes in the immune response to determining cell fate and tissue development, mechanical forces play a key regulatory role throughout biology. This emergent field of "mechanobiology" is leading to new understandings of disease such as bleeding disorders, cancer, osteoporosis and asthma, as we recognize that mechanics can play a large role in physiological responses at the molecular, cellular and organismic levels. Technological developments that enable precise manipulation of single molecules and cells (e.g. optical tweezers and AFM) have been a driving force in the development of the field. However, growth of the field is impeded by limited access to such technology as it can be expensive, technically challenging, and low-throughput. To overcome these challenges, we will develop a bold approach for applying controlled forces to microscopic samples with single-molecule precision that is inexpensive, simple to use, and high-throughput. By integrating a centrifuge and a microscope, we have demonstrated a prototype Centrifuge Force Microscope (CFM) to perform thousands of single-molecule force experiments in parallel. We propose to take this concept of single-molecule centrifugation to the next level, by developing a powerful, multi- purpose, benchtop instrument for mechanobiology that can be used by a variety of biomedical researchers. We will accomplish this by 1) Enabling massively parallel single-molecule measurements of structural transitions such as protein unfolding by integrating high-resolution imaging into the CFM; 2) Increasing the accessibility of single-molecule manipulation techniques by miniaturizing the high-resolution CFM to be incorporated directly into a standard benchtop centrifuge; 3) Demonstrating the versatility of this CFM by measuring intra- and inter-molecular bond strengths at both the single-molecule and single-cell levels. In summary, this project will result in an accessible, high-throughput and high-resolution new platform for measuring biological systems under mechanical force at the nanoscale with a broad range of applications, ranging from measuring structural transitions within individual molecules, to measuring the affinity of single cells, to measuring the compliance of soft samples. This instrument will dramatically reduce cost and improve performance and safety by leveraging one of the most common pieces of laboratory equipment - the benchtop centrifuge. This approach will remarkably lower the barrier for researchers to do single- molecule manipulation experiments by requiring little technical expertise and by offering a 1000 fold efficiency boost and a 10-100 fold cost improvement from many other methods. This project is significant since it will open up the fields of mechanobiology and single-molecule manipulation to many new researchers and systems, accelerating the pace of discovery.